There have been several major accomplishments within the past fiscal year. First, we identified a homolog of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The Capitella teleta AChBP (ct-AChBP) has 21-30% amino acid identity with known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened cys-loop compared to other cys-loop receptors, and a variation on a conserved C-loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D-loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand-binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to homologs with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and &#61537;-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 uM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (KD = 163 uM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may possess variation in hallmark motifs such as ligand-binding residues and cys-loop length, and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca. Second, we used voltammetric analyses to show that low (100-500 nM) doses of nicotine regulate striatal dopamine by inhibiting release evoked by a single stimulation to a greater extent than release evoked by high frequency stimulations. This frequency-dependent inhibition is due to nicotine desensitizing heteromeric &#946;2 subunit-containing nicotinic acetylcholine receptor (nAChR) subtypes. Surprisingly, a high dose of nicotine (2 M;capable of interacting with additional nAChR subtypes) produced an inhibition of dopamine evoked by high frequency stimulation, an effect that was not seen with the low dose of nicotine or the &#946;2 antagonist, DH&#946;E. This inhibition was replicated by application of &#945;7 nAChR antagonists MLA or &#945;-bungarotoxin in conjunction with the low dose of nicotine or DH&#946;E. Blocking &#945;7 receptor function alone produced a modest increase in dopamine evoked by single pulse stimulation while not affecting dopamine evoked by high frequency stimulation. The antagonist results were mimicked using selective &#945;7 agonists PHA 543613 and PNU 282987. The frequency dependence of the low dose nicotine inhibition therefore requires functional &#945;7 nAChRs, and may arise from differing levels of endogenous ACh evoked by the stimulation. Third, we continued to explore how various residues in the extracellular domain affect desensitization of nAChRs. The rat &#945;7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the beta9 strand. The substitution of this proline residue at position 180 (P180) to either threonine (&#945;7-P180T) or serine (&#945;7-P180S) slowed the onset of desensitization dramatically, with half-times of 930 and 700 msec, respectively, compared to 90 msec for the wildtype receptor. To investigate the importance of the hydroxyl group on the position 180 sidechains, the mutant receptors &#945;7-P180Y and &#945;7-P180F were studied and showed half-times of desensitization of 650 and 160 msec, respectively. While a position 180 sidechain OH group may contribute to the slow desensitization rates, &#945;7-P180S and &#945;7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H-bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the &#945;7-P180T receptor, there was a significantly reduced closed time without any change in single channel conductance (as compared to wildtype). Kinetic simulations indicated that all changes observed for the mutant channel behavior were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular Dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer beta-sheet that occurs when the P180 residue is mutated. All mutations analyzed increased about twofold the predicted number of H bonds between the residue at position P180 and the backbone of the beta10 strand. Moreover, the &#945;7-P180T and &#945;7-P180S mutations also formed some intrastrand H bonds along the beta9 strand, although H bonding of the OH groups of the threonine or serine sidechains was predicted to be infrequent. Our results indicate that rapid desensitization of the wildtype rat &#945;7 nAChR is facilitated by the presence of the proline residue within the beta9 strand.